Necrotic Enteritis (NE), caused by Clostridium perfringens, is a debilitating disease that leads to poor quality of life in broiler chickens and results in production losses to the poultry industry. Traditionally, antibiotics have been used to inhibit development of necrotic lesions in the gut of affected chickens. Due to the possibility of antibiotic resistant strain development, alternative methods to control this disease are required. In previous studies, NetB, the key virulence factor, and Alpha-toxin, one of the secondary virulence factors, have been used as subunit vaccines individually with only partial protection induced. A literature review, and bioinformatic analysis of the C. perfringens genome led to the identification of additional putative virulence factors and vaccine candidates. A multi-valent (MV) fusion protein consisting of fragments from four different virulence factors was designed, and recombinant protein produced. The MV-protein vaccine and each of the individual proteins were tested in a necrotic enteritis challenge model. Birds were vaccinated twice then challenged in-feed with a pathogenic strain of C. perfringens and necrotic lesion scored. The birds vaccinated with the MV-protein showed a 95% reduction in lesions compared to birds vaccinated with individual proteins or adjuvant alone (30%). Titres of specific IgY- induced by the MV protein were significantly higher than those for the individual proteins. Western blot and ELISA results showed that vaccination with MV-protein induced antibodies specific for all four target protein fragments that made up the MV-protein. The MV-protein vaccine induced specific antibody responses and provided significant protection against the formation of necrotic lesions and provides the basis for ongoing vaccine development.